ESSENTIALS OF GLYCOBIOLOGY

LECTURE 15

The O-GlcNAc Modification

Hud Freeze

“Dynamic Interplay Between O-GlcNAc and O-Phosphate: Roles in Transcription,

Signaling and in Cellular Response to Stress.”

Gerald W. HartJohns Hopkins University

School of Medicine

ADAPTED FROM

O-GlcNAcO-GlcNAc O- Linked N-AcetylglucosamineO- Linked N-Acetylglucosamine

Tyr-Ser-Pro-Thr-Ser-Pro-Ser

O

AcNHHO

OH

O

O

AcNHHO

HO

OH

O

HO

A dynamic post-translational modification

O-GlcNAc Transferase O-GlcNAcase

Key Features of O-GlcNAc :Key Features of O-GlcNAc :NOTNOT elongated to more complex elongated to more complex structures.structures.Localized to the cytoplasm and nucleus.Localized to the cytoplasm and nucleus.Present in all higher eukaryotes studied.Present in all higher eukaryotes studied.As abundant as phosphorylation; As abundant as phosphorylation; UDP-GlcNAc is Nearly as abundant as ATP.UDP-GlcNAc is Nearly as abundant as ATP.O-GlcNAc proteins are also O-GlcNAc proteins are also PhosphoproteinsPhosphoproteinsO-GlcNAc and Phosphorylation are often O-GlcNAc and Phosphorylation are often reciprocal.reciprocal. Highly dynamic modification - a Highly dynamic modification - a regulatory role.regulatory role.

Non-CapsidViral

Proteins

Nuclear PoreProteins

RNA Polymerase II& Transcription Factors

Tumor Suppressors& Oncogenes

Cytoskeletal Proteins

ChromatinProteins

Protein TranslationRegulatory Factors

Cytoplasmic Face ofVesicle Proteins

ParasiteProteins

CytosolicEnzymes

Cytoplasmic Tailsof Membrane Proteins

RNA ProcessingProteins

ProteosomalProteosomalProteinsProteins

pI 4 pI 9 pI 4 pI 9

Unbound sWGA bound--GlcNAc Eluted

-110-

MW

-20-

Silver stain of 80 ug total protein from each fraction(starting material 30 mg nucleocytoplamic lysate from HeLa cells)

Enrichment of O-GlcNAc Modified Proteins using sWGA:

pI: 3 --> 10

2nd Dimension:10% SDS-PAGE

Visualization:Silver Stain

CTD 110.6 MAb Immunopurifies Many Glycoproteins From HeLa Cell Cytoplasmic and

Nuclear Extract:

Nuclear Pore ProteinsNuclear Pore Proteins - - p62, Nup54, 155, 180, 153, 214, 358; p62, Nup54, 155, 180, 153, 214, 358; Chromatin ProteinsChromatin Proteins - Many- Many

Transcription Factors - Transcription Factors - SP1, cfos, cJun, CTF, HNF1, v-ErbA, Pancreas Specific TF, SRF, c-Myc, p53, Estrogen Receptors, ß-catenin, NFKB, ELF-1, PAX-6, Enhancer Factor D, Human C1, Oct1, plakoglobin, YY1, PDX-1, CREB, Rb, p107, RNA Polymerase II.

RNA Binding Proteins - HnRNP-G, Ewing Sarcoma RNA binding Protein, EF4A1, EF1alpha, 40S ribosomal s24, many ribosomal proteins.

Phosphatases/Kinases/Adapters nuclear Tyr phos’ase p65, CKII, IRS-1,2, GSK3ß, PI3-kinase

Cytoskeletal Proteins - cytokeratins 8, 13, 18, Neurofilaments H, M, L, Band 4.1, talin, vinculin, ankyrin, synapsin I, myosin, E-cadherin, cofilin, tau, Maps 2, 4, Dynein, alpha tubulin, AP3, AP180, ß-APP, ß-synuclein, piccolo.

Chaperones - HSP27, alpha crystallins, HsC70, HsP70, HsP90 Metabolic Enzymes - eNOS, Enolase, Glyceraldehyde-3-phosphate dehydrogenase,

phosphoglycerate kinase, pyruvate kinase, UDP-glucose synthase, glycogen synthaseOther Regulatory Proteins - Eukaryotic peptide chain initiation factor 2 p67, OGT,

CRMP-2, Ubiquitin carboxy hydrolase (UCH), Glut 1, Annexin 1, Nucleophosmin, proteasome components C2, 5/9 and 9/14 of 26S proteasome regulatory & catalytic subunits, respectively, Q04323 UCH homolog, Sec23, Ran, peptidyl prolylisomerase, Rho GDP-dissociation inhibitor, GABA Receptor interacting protein 1,

Viral Proteins - adenovirus fiber, SV40 Large T Ag;, Baculovirus Teg protein,

Some Identified O-GlcNAc-Modified Proteins:Some Identified O-GlcNAc-Modified Proteins:

Some Clues To O-GlcNAc Functions:

Required for Life at Single Cell Level - (OGT -/+ still emb. lethal.) Regulates Transcriptional Activation or Suppression, Dep. upon Metabolism - SP1,

ERs, Stat5, NFkB, p53, c-Myc; Regulates Degradation. Regulates Protein Synthesis (via p67-EIF2 kinase);(Gupta & Datta) Regulates ß-catenin and E-cadherin trafficking (Andrews et al.) Neurodegenerative Disease - Tau, ß-APP, NFs, O-GlcNAcase maps to late-AD locus;

OGT Maps to Parkinson Dystonia Locus; O-GlcNAc is Reduced in Human AD. YY1 Transcription Factor is Regulated by O-GlcNAc - Prevents binding to Rb. O-GlcNAc on Glycogen Synthase Prevents its Activation by Insulin (Parker; McClain). O-GlcNAc on eNOS prevents activation by Akt (Brownlee) Retinoblastoma (Rb) is O-GlcNAc Modified in G1 & O-GlcNAc-Rb binds E2F. O-GlcNAcylation of 26S Proteosome Inhibits Degradation; 5/19 and 9/14 of Catalytic

Core and Reg. Core subunits, respectively, are modified (Cell 115, 715; BBRC 312,1284) Regulator of Gibberellic Acid Signaling in Plants-OGT=Spy; Secret Agent

Blocks Insulin Signaling and OGT Over-Expression in Muscle or Adipose Causes Diabetes in Mice (McClain & Hanover)

(>~200 Papers Since 1984 Directly Concerned with O-GlcNAc)(>~200 Papers Since 1984 Directly Concerned with O-GlcNAc)

SiteA

SiteB

SiteA

SiteB

SiteA

SiteB

SiteA

SiteB

OO

OH

HOHO

HNAc

SiteA

SiteB

OPO3-

OPO3-

OO

OH

HOHO

HNAc

OO

OH

HOHO

HNAc

1

2

3

4

5

6

7

Complex Interplay Between O-Phosphate & O-GlcNAc:Complex Interplay Between O-Phosphate & O-GlcNAc:

QuickTime™ and aGIF decompressor

are needed to see this picture.

OPTIONS FOR O-GlcNAc MODIFICATION

O-GlcNAc Transferase Summary:

OGT has been highly conserved throughout evolution.

OGT is a unique glycosyltransferase. Localized near centromere on X-chromosome. 1 Normal Allele is Required for ES Cell Viability

(Marth et al.)

OGT structure

catalytic

TPR domains - (tetratricopeptide repeats)

Generates O-GlcNAc linkage on peptides

-Single gene encoding103 kDa peptidemigrates at 110 kDa

Inexact peptide sequence motif for glycosylationAssociates with self and other proteins in complex

Expressed in all mammalian tissues studiedLocated in nucleus and cytoplasm

Modified by O-GlcNAc and tyrosine-phosphate

O-GlcNAc O-GlcNAc

TPRDomain

CatalyticDomain

TPR InteractingEffector Proteins

OGT mRNAOGT Splice

Variants

ProteolyticProcessing OGT Variant

O-GlcNAc O-GlcNAc O-GlcNAcO-GlcNAcO-GlcNAc

O-GlcNAc Modified Proteins

OGT

Multimerization

[UDP-GlcNAc][UDP]

SubstrateSelectivity

Post-translationalModifications

TranscriptionalRegulation

RNA Processing

Identified OGT-TPR Binding Proteins:

Co-Repressor mSin3A binds OGT and Suppresses SP1 Driven Transcription (Kudlow)

GRIF-1 - Targets to GABA Receptor (Anne Stephenson) and Regulates its signaling; OGT binding protein (Sai Iyer).

Milton (Drosophila GRIF-1 analog) required for kinesin-mediated axonal transport of mitochondria to Synapses (Neuron 36, 1063 (Schwarz).

OIP106 - GRIF-1-Like Protein Targets OGT to RNA polymerase II - binds via TPRs.

Many more yet to be identified - pulled out by yeast 2H.

(Proteins That Target OGT and Control Specificity)(Proteins That Target OGT and Control Specificity)

Effect of [UDP-GlcNAc] On OGT Activity In the Presence of Alkaline Phosphatase

0

200

400

600

800

1000

1200

1400

0 10 20 30 40 50

y = 0.6802 + 27.714x R= 0.99833

pmol

inco

rpor

ated

[UDP-GlcNAc] mM

50 mM!!50 mM!!

PhysiologicalPhysiologicalRangeRange

O-GlcNAc’ase Structure

OGA

Expressed in all human tissues studied

OGA peptide cleaves GlcNAc from glycopeptides

Single gene encodes 916 amino acid polypeptide of 103 kDa migrates at 130 kDa

Predominantly expressed in the cytoplasm

Highly conserved in mammals and found in C. Elegans

Located on Chromosome 10 in humans

Functional aspects of O-

GlcNAcase Maps exactly to 10q23.1 - late onset

Alzheimer’s Disease Locus. Ogase has Histone Acetyltransferases

activity.Inhibition BLOCKS insulin signaling.

53 nM Ki inhibitor of53 nM Ki inhibitor ofO-GlcNAcase.O-GlcNAcase.

Identification of O-GlcNAc Modified

Proteins by Mass Spectrometry

Generally Generally Not Not Affect Gel Affect Gel Electrophoresis Electrophoresis Not Easily Labeled - No Not Easily Labeled - No 3232P!P!Very LabileVery Labile - both Chemically - both Chemically and Enzymatically- Falls Off in and Enzymatically- Falls Off in MS.MS.StoichiometryStoichiometry Similar to O- Similar to O-Phosphate.Phosphate.

Why Did O-GlcNAc Remain Undetected?Why Did O-GlcNAc Remain Undetected?

Fragmentation of GlcNAc-CTD by CID-MS/MS Parent ion 535 ([M+2H+] + 1 GlcNAc)

200 300 400 500 600 700 800 900 1000 1100 1200

m/z

05

101520253035404550556065707580859095

100

Rel

ativ

e A

bund

ance

866.3

410.3 819.2203.8616.2

433.8250.9476.8

NL 7.62e6Y--S--P--T--S--P--S--K

b ions - 164 251 348 449 536 633 720 848

866 703 616 519 418 331 234 147 - y ions

O-GlcNAc (204)

848.4

GlcNAc

b2

y6

b8

y8

Poor FragmentationPoor Fragmentation

No Site Information.No Site Information.PeptidePeptide

sugarsugar

Alkaline inducedElimination

CNH

C

O

CH2

(Serine-O-GlcNAc)(dehydroalanine)

CNH

C

O

CH2

OGlcNAc

H

Strategy for O-GlcNAc/O-Phosphate site mapping

0

100

0

100

0

100

1314.3

PSVPVSerGSAPGR

O-GlcNAc

PSVPVSerGSAPGR

DTT

PSVPVSerGSAPGR

BAP

1247.4

1421.7

m/z1100 1650

m/z1100 1650

m/z1100 1650

MALDI-TOF

Replacement of O-GlcNAc with DTTUsing -elimination/michael addition

CNH

C

O

CH2H

DTT (or BAP)

Michael Addition

HSCH2CHOHCHOHCH2SH (DTT)

1. Provides tag forAffinity enrichment

2. Tag is stable in massspectrometer

Relative intensity

Relative intensity

Relative intensity

BEMAD Useful for Simultaneous Mapping of O-GlcNAc and O-Phosphate:

O-GlcNAc much more sensitive to ß-Elimination. Treatment with Phosphatase & O-GlcNAcase. Density-Labeled DTT is Cheap & Available. Same Approach work for ‘classical’ O-glycans.

Phosphorylation Mapping studies Must Account for Abundance of O-GlcNAc.

QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.

Make GlcNAc derivative with a N3-reactive azide instead of AcUDP-GlcN -->-->Protein with O-GlcN

Probe with FLAG or biotin

Detect Probe

Another way to recognize O-GlcNAc proteins

Vocadlo DJ, Hang HC, Kim EJ, Hanover JA, Bertozzi CR.Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9116-21

OH

O

AcNHHO

HOO

-Leu-Leu-Pro-Thr58-Pro-Pro-Leu-

-Leu-Leu-Pro-Thr58-Pro-Pro-Leu-OO

O O

Reciprocal Reciprocal O-GlcNAcylation & Phosphorylation of O-GlcNAcylation & Phosphorylation of c-Mycc-Myc::

-P-O-P-O----OO

Thr58 is Mutation Hot Spot Thr58 is Mutation Hot Spot In Human Lymphomas.In Human Lymphomas.

Mutations of c-Myc in LymphomasMajor O-GlcNAc Site.Major O-GlcNAc Site.

Transactivation DomainTransactivation Domain

GSK3GSK3

ErkErk

Site-Specific mAB - Thr58-O-GlcNAc:1. Prevention of Phosphorylation at Ser 62 Elevates O-GlcNAc at Thr 58.2. Stim. Of Growth Reduces O-GlcNAc at T58; Increases Phos. & vice versa.3. Inhibition of GSK3ß increases O-GlcNAc at T58.4. Regulates c-Myc association with Tumor Suppressor Rb p107.

Heat ShockUV

Osmotic StressFree Radicals

Reductive Stress

Heat Shock ProteinsGlutathione

SODCatalase

X X

O-GlcNAc O-GlcNAc

The rapid increase in O-GlcNAc and the effect of increased HSP70 in the presence of increased O-GlcNAc suggests that O-

GlcNAc forms an integral component of the cell’s stress signaling pathways.Natasha ZacharaNatasha Zachara

The Addition of O-GlcNAc to Proteins in Response to Stress is Dynamic

Cos-7 cells were treated with heat shock (45oC, 1h) and allowed to recover at 37oC.

177

114

80

64

50

37

WB: O-GlcNAc

WB: HSC/HSP70

1 3 6 48 48 hTime post-heat shockCont Cont

9 241

O-GlcNAc Precedes HSP70 O-GlcNAc Precedes HSP70

O-GlcNAc is A Sensor of Cellular Stress:

O-GlcNAc and OGT are rapidly elevated in response to many forms of cellular stressThis is dose dependent; does not require mRNA or Protein

Synthesis Increased levels of O-GlcNAc results in increased

thermotolerance In part a result of more rapid increased HSP70

production/stabilization.HSP70 as an O-GlcNAc lectin - 2 French Groups.Decreased protein aggregation?

Decreased O-GlcNAc Results in decreased thermotolerance

UDP-GlcNAc as a Metabolic Sensor:

UDUDPP--GlcGlcNNAcAc

Glucose Glucose MetabolismMetabolism

NitrogenNitrogenMetabolismMetabolism

Fatty AcidFatty AcidMetabolismMetabolism

Nucleotide Nucleotide MetabolismMetabolism

OverallOverallEnergy Energy

REVIEW OF METABOLIC PATHWAYS

Elevation of O-GlcNAc Blocks Insulin Signaling:Elevation of O-GlcNAc Blocks Insulin Signaling:• Huge Literature - Diabetes Requires Glc to GlcNAc.Huge Literature - Diabetes Requires Glc to GlcNAc.• GlcN 10X more potent than Glc in inducing Insulin-Resistance.GlcN 10X more potent than Glc in inducing Insulin-Resistance.• O-GlcNAc is Elevated in Muscle and Adipose in Diabetic Animals.O-GlcNAc is Elevated in Muscle and Adipose in Diabetic Animals.

[UDP-GlcNAc]

HSP

OGT

x-Ser/thr-x-x x-Ser/thr-x-xO-GlcNAc

O-GlcNAcase

glucoseGlut4vesicle

PDK1

AKT1/2

Thr 308-P

PKC

GSK3

P-Y IRS1/2

Insulinreceptor

P-Y

p85

p110

PI 3-kinase

Glycogen synthesis

Ser 9-P

PUGNAc

!Insulin resistance

Elevation of O-GlcNAc Blocks Insulin Elevation of O-GlcNAc Blocks Insulin Signaling:Signaling:

• Huge Literature - Diabetes Requires Glc to GlcNAc.Huge Literature - Diabetes Requires Glc to GlcNAc.• GlcN 10X more potent than Glc in inducing Insulin-Resistance.GlcN 10X more potent than Glc in inducing Insulin-Resistance.• O-GlcNAc is Elevated in Muscle and Adipose in Diabetic O-GlcNAc is Elevated in Muscle and Adipose in Diabetic Animals.Animals.

****

**??

Does Specific Elevation of Does Specific Elevation of O-GlcNAc Cause InsulinO-GlcNAc Cause InsulinResistance? Resistance? Yes!Yes!

**

**

Glucosamine (mM) 0 0 5 5chronic insulin (nM) 0 1 1 0

Deo

x ygl

ucos

e u p

tak e

(in

sulin

de p

end e

nt 14

[C] D

PM)

20000

10000

0

51%

WB: anti-O-GlcNAc

Insulin Resistance induced through the HBP correlates with increased O-GlcNAc in 3T3-L1 Adipocytes

Requires Both GlcN & InsulinRequires Both GlcN & Insulinfor Insulin-Resistancefor Insulin-Resistance& for Elevated O-GlcNAc.& for Elevated O-GlcNAc.

(A)

Insulin concentration (nM)

23%

39%

Acute insulin (nM) 0 0 1 1 10 10PUGNAc - + - + - +

WB:anti-O-GlcNAc110.6 antibody

PUGNAc - + - + - + - + - + Acute Insulin (nM) 0.1 0.33 1 3.3 10

Deo

xygl

ucos

e u p

tak e

(in

sulin

de p

end e

nt 14

[C] D

PM)

(B)

PUGNAc Elevates O-GlcNAc and Induces

Insulin Resistance in 3T3-L1 adipocytes:

53 nM Ki inhibitor of53 nM Ki inhibitor ofO-GlcNAcase.O-GlcNAcase.

Insulin Signaling Pathway for Glut4 Translocation to Plasma Membrane - Where is O-GlcNAc

Blocking?

IRS

PI3K

PDK1

AKT

GSK3

PKC

Glut4

???

IR

-O-GlcNAc-O-GlcNAc

Many O-GlcNAcMany O-GlcNAcIncluding Including Munc18Munc18

Glycogen SynthaseGlycogen Synthase-->-->

-O-GlcNAc-O-GlcNAc

-O-GlcNAc

-O-GlcNAc

PUGNAc does PUGNAc does notnot effect the protein levels or insulin stimulated effect the protein levels or insulin stimulated tyrosine phosphorylation of the insulin receptor or IRS-2.tyrosine phosphorylation of the insulin receptor or IRS-2.

PUGNAC PUGNAC BlocksBlocks Insulin-Stimulated Insulin-Stimulated Phosphorylation of Phosphorylation of Threonine 308Threonine 308 on AKT/PkB*: on AKT/PkB*:

AKT Activation is Blocked!AKT Activation is Blocked!

PUGNAc PUGNAc BlocksBlocks Insulin-Stimulated Phosphorylation of GSK3ß at Insulin-Stimulated Phosphorylation of GSK3ß at Ser9Ser9, , But But NotNot Thr202/204 MAPK or Ser473 on Akt/PKB: Thr202/204 MAPK or Ser473 on Akt/PKB:

GSK3ß Activation is Blocked!GSK3ß Activation is Blocked!

[UDP-GlcNAc]

HSP

OGT

x-Ser/thr-x-x x-Ser/thr-x-xO-GlcNAc

O-GlcNAcase

glucoseGlut4vesicle

PDK1

AKT1/2

Thr 308-P

PKC

GSK3

P-Y IRS1/2

Insulinreceptor

P-Y

p85

p110

PI 3-kinase

Glycogen synthesis

Ser 9-P

PUGNAc

!Insulin resistance

Elevation of O-GlcNAc Blocks Insulin Signaling:Elevation of O-GlcNAc Blocks Insulin Signaling:•Blocks AKT phos. at T308 and S9 on GSK3ßBlocks AKT phos. at T308 and S9 on GSK3ß

•Inhib. OGase greatly increases OG on ß-catenin Inhib. OGase greatly increases OG on ß-catenin and IRS1.and IRS1.

Transgenic Mice with OverexpressedTransgenic Mice with OverexpressedOGT in Muscle or Adipose - OGT in Muscle or Adipose - BecomeBecome

Diabetic.Diabetic. (McClain & Hanover) (McClain & Hanover)

****

**??

**

??

Insulin (nM) 0 1 100

IP: OGTWB: OGT

IP: OGTWB:PY

OGT

OGT

Fig. Insulin stimulated tyrosine phosphorylation of O-GlcNAc transferase (OGT). 3T3L1 adipocytes starved of growth factorsFor 16 hours were stimulated 10 minutes with insulin at suboptimal (1 nM) or optimal (100 nM) concentrations, and OGT Immunoprecipitates were western blotted for either OGT levels or phosphotyrosine.

Note: activity of these immunoprecipWas tested with no differences against CKII peptide. Post-translational modification may effectlocalization, substrate specificity, binding Partners, ect. (SH2 domain containing protein?)Phospho-tyrosine response would place OGT in Signaling pathway downstream of insulin Receptor. time point coincides with start of glucose Uptake assay.

Insulin Stimulates (10 Insulin Stimulates (10 min)Tyr-min)Tyr-PP

Of O-GlcNAc Transferase:Of O-GlcNAc Transferase:

OGT is Directly in the IR Signaling Pathway.OGT is Directly in the IR Signaling Pathway.

Fig. Insulin stimulates dynamic O-GlcNAcylation in 3T3L1 adipocytes. 3T3L1 adipocytes starved of growth factors for 16 hours Were stimulated with 100 nm insulin for 10 minutes, and whole cell lysates were separated by 2D gel electrophoresis and western blotted With an anti-O-GlcNAc specific antibody.

Spot # response to insulin1 decreased O-GlcNAc2 decreased O-GlcNAc3 likely phosphorylation shift4 decreased O-GlcNAc5 likely phosphorylation shift6 decreased O-GlcNAc7 decreased O-GlcNAc

1

1

2

2

3

3

4

4 5

5

6

6

7

7

pI 4.5pI 5

unstimulated

Insulin stimulated(100 nM for 10 min.)

Proteomics Approach to O-GlcNAc & Insulin SignalingProteomics Approach to O-GlcNAc & Insulin Signaling::Insulin Insulin Rapidly DecreasesRapidly DecreasesO-GlcNAc on Many Proteins:O-GlcNAc on Many Proteins:

Insulin Causes Major Insulin Causes Major Changes in O-GlcNAc Changes in O-GlcNAc in in 10 min.10 min.

(Small Portion of (Small Portion of Gel Shown)Gel Shown)

Conclusions:• O-GlcNAc is a Major Regulatory PTM in all multicellular

eukaryotes - Plants & Animals.• O-GlcNAc Accounts for Many of the Biological Affects

Attributed Hexosamine Biosynthetic Pathway - Diabetes & Glucose Toxicity.

• O-GlcNAc is Required for Life at the Single Cell Level.• O-GlcNAc is as abundant as Phosphorylation and

Often Competes with it.• O-GlcNAc - “Metabolic Sensor” to Modulate Signaling

& Transcription in Response to Cellular Status.• Many Toxic Effects of Hyperglycemia Likely Result

From Dysregulation of the Balance Between O-GlcNAc and Phosphorylation.